WO2016111538A2

WO2016111538A2 - Reagent kit for detecting sex hormone and method for detecting sex hormone using same

Info

Publication number: WO2016111538A2
Application number: PCT/KR2016/000088
Authority: WO
Inventors: 주재범; 전향아; 왕루이; 이상엽; 윤영호; 홍성현
Original assignee: 한양대학교 에리카산학협력단; 의료법인 이원의료재단
Priority date: 2015-01-09
Filing date: 2016-01-06
Publication date: 2016-07-14
Also published as: US20220178952A1; WO2016111538A3

Abstract

The present invention provides a reagent kit for detecting a sex hormone and a method for detecting a sex hormone using the same, the reagent kit comprising: a first reagent comprising a sex hormone and a metal nanoprobe in which a Raman reporter is immobilized; and a second reagent comprising magnetic particles in which an antibody for detecting the sex hormone is immobilized.

Description

Reagent kit for detecting sex hormones and method for detecting sex hormones using the same

The present invention provides a reagent for detecting sex hormones based on Surface-Enhanced Raman Sacattering (hereinafter referred to as 'SERS'); A method of detecting sex hormones using this reagent; And it relates to a method for diagnosing precocious puberty using the above method.

Puberty normally appears at an average of 10-11 years old in Korean girls and 13-14 years old in boys. At 15 years old in girls and 18 years old in boys, sexual maturity reaches adult levels and growth stops.

In general, precocious puberty refers to cases in which puberty occurs in girls younger than 8 years old and younger than 9 years old. In girls only Breast development before age 8, and in boys, testicles before age 9 are said to be precocious puberty.

Recently, precocious puberty has emerged as a social issue. The prevalence of precocious puberty is known to be largely due to the increasing effects of environmental hormones and obesity in children.

When precocious puberty occurs when mental maturation cannot keep up with physical development, young children may be stressed, and the growth plate may close prematurely, causing serious problems that can stop height growth. Therefore, there is a need for diagnosis and / or treatment of precocious puberty.

According to a recent analysis by the Korea Health Insurance Review & Assessment Service, precocious pneumonia patients more than tripled from 21,1712 in 2009 to 66,395 in 2013. appear. Most AD patients can be improved with hormonal therapy, but getting the right diagnosis is the most important. In other words, it is necessary to diagnose a child's precocious puberty early.

The typical sex hormone associated with precocious puberty is estradiol. Normally, the estradiol content in women is above 60 pg / mL, but in men, postmenopausal women, and pre-pubertal children, it is generally below 10 pg / mL.

Testosterone is present in adult males at 4.04 to 7.21 ng / mL, adult females at 0.37 to 0.81 ng / mL, young boys below 2.54 ng / mL, and young girls below 0.20 ng / mL. Plasma testosterone levels vary more than 1,000-fold depending on age, sex and disease.

Existing estradiol or testosterone assays include, i) immunoassay by chemiluminescence, ii) radioactivity based immunoassay, and iii) high performance liquid chromatography mass spectrometry (LC / MS / MS). i) Immunoassay by chemiluminescence (ELISA Kit) is a test method using color change, and has the advantage of using high sensitivity and simple operation method. However, if the limit of detection (LOD) is 30-100 pg / mL and the concentration of estradiol (or testosterone) in the sample is low, it is impossible to measure and a lot of diagnostic errors. ii) Estradiol radioimmunoassay kit is a method of measuring the amount of estradiol (or testosterone) detected by radioactivity. The detection limit is 10 pg / mL and has a high sensitivity. However, when the concentration of estradiol (or testosterone) in the sample is low, the accuracy is very poor and there is a disadvantage that the sample may be contaminated by radiation exposure. iii) The high performance liquid chromatography mass spectrometry has the advantage that the measurement method is accurate. However, due to the complex examination method, the long detection time, and the high cost of the test, it is not suitable for use as a diagnostic method in the department of diagnostic laboratory medicine.

Automated immunoassay using chemiluminescence is widely used for diagnosis of precocious puberty in most of the university hospitals. Typical commercial equipment includes Abbott Architect, Beckmann, Roche Covas, Siemens ADVIA Centaur, Tosoh ST and Vitros. However, currently available diagnostic equipment measurements are available for estradiol concentrations ranging from 50-200 pg / mL, as shown in Table 1 below (Source: College of American Pathologists, www.cap.org/Estradiol Information). It has large standard deviation (SD) and coefficient of variation (CV) values, and concentrations of estradiol below 10 pg / mL cannot be detected. Therefore, current immunodiagnostic techniques cannot accurately diagnose precocious puberty, and there is no gold standard method for diagnosing it.

TABLE 1

Non Patent Literature 1 (William Rosner, et al., 2013. J Clin Endocrinol Metab , 98 (4) 1376-1387, describes in FIG. 1 that the concentration of estradiol below 10 pg / mL is currently difficult to detect. In addition, in Non-Patent Document 1, in order to monitor female breast cancer patients treated with hormone inhibitors (aromatase inhibiotors), the estradiol concentration is usually 10-15 pg / mL before treatment, and is maintained at 1 pg / mL or lower after treatment. It is important to note that accurate determination of estradiol at low concentrations is important (see right column on page 1379).

As described above, to date, there is no accurate method for measuring sex hormones such as estradiol or testosterone concentrations below 10 pg / ml. In addition, after hormone therapy in precocious puberty children, sex hormone concentrations must be suppressed and maintained at low concentrations, so accurate sex hormone concentration measurement at low concentrations (less than 10 pg / ml) is necessary. Clinicians need to provide accurate sex hormone concentration information in patients to determine the effectiveness of drug treatment.

In addition, it is emphasized that such a study is very necessary since no suitable sex hormone detection method for diagnosing precocious puberty of children has been studied (see the right column on page 1380 of Non-Patent Document 1). In addition, Non-Patent Document 1 strongly insists on the need for a measurement method capable of measuring very low concentrations of estradiol (see the conclusion section on page 1384 of Non-Patent Document 1).

In Non-Patent Document 2 (Genna Rollins, May 2013, Clinical Laboratory News, Vol. 39, No. 5) published by the American Society for Diagnostic Medicine, “ The current platform Assays can't distinguish between 10 and 60 pg / mL. ” , revealing the limitations of current precocious puberty diagnostics. Non-Patent Document 2 emphasizes the need to develop a new technology that can accurately and reliably quantify estradiol at a concentration of 10 pg / mL or less and apply it to routine clinical diagnosis. Through the non-patent document 2, it can be seen that the currently required technology, like the title of the non-patent document 2, is “ A Call for Better Estradiol Measuremen t”. Specifically, a new sex hormone detection method that can reduce the sex hormone diagnosis time, significantly increase the sensitivity and accuracy, and can be applied to the diagnostic reagent-based automated immunoassay, which is performed in the diagnostic test medicine, and the standard of sex hormone using the same Development of measurement technology for gold standard method is urgently needed.

Surface-enhanced Raman scattering (SERS) based detection method is an assay that can overcome the detection sensitivity limit of Raman spectroscopy. This assay is a way to quantify target material by measuring the change in intensity of amplified characteristic SERS peaks of Raman reporter molecules. When reporter molecules are adsorbed on rough metal surfaces and exposed to excitation light (laser light), electromagnetic and chemical enhancement occurs at the SERS active site of the reporter molecule, known as “hot junction,” resulting in a significant increase in the SERS signal. (Non-Patent

Documents

3, 4, and 5). This augmentation effect is expected to solve the problem of low sensitivity, which is a disadvantage of the conventional Raman detection method, and is expected to exceed the accuracy and detection limits of the conventional chemiluminescence assay and radioactivity-based immunoassay.

The present inventors have continued to research to overcome the above-mentioned limitations of the prior art. As a result, it is possible to reliably analyze sex hormones with high sensitivity using a new concept of nanoplasmonics-based SERS, which is different from radioimmunoassay, enzyme immunoassay and chemiluminescence analysis. A new sex hormone immunoassay has been developed. The present invention is evaluated as a new concept of sex hormone diagnosis technology that can overcome the problems (detection sensitivity and accuracy) of the diagnosis of precocious puberty through the detection of existing sex hormones.

(Non-Patent Document 1) William Rosner, et al., April 2013. J Clin Endocrinol Metab , 98 (4) pp. 1376-1387

(Non-Patent Document 2) 2. Genna Rollins, May 2013, Clinical Laboratory News, Vol. 39, No. 5

(Non-Patent Document 3) 3. Kneipp, J. et al., 1997. Phys. Rev. Lett . 78, pp. 1667-1670

(Non-Patent Document 4) 4. Nie, SM and Emory, SR, 1997. Science 275, pp. 1102-1106

(Non-Patent Document 5) 5. Kneipp, J. et al., 2006. Nano Lett . 6 (10), pp. 2225-2231

An object of the present invention is to provide a reagent kit for detecting sex hormones.

Another object of the present invention is to provide a method for detecting sex hormone based on Surface-Enhanced Raman Scattering (SERS).

Another object of the present invention is to provide a method for diagnosing precocious puberty using the sex hormone detection method.

This specification refers to a number of papers and citations and indicates the citations. The contents disclosed in the cited papers and patent documents are incorporated in their entirety as part of the contents of the present specification to more clearly describe the technical level of the present invention and the contents of the present invention.

The present invention includes a first reagent comprising a metal nanoprobe to which sex hormones and Raman reporters are immobilized; And

A second reagent comprising magnetic particles immobilized with the sex hormone detection antibody;

It provides a reagent kit for detecting sex hormone comprising a.

1 schematically shows a first reagent and a second reagent of the present invention.

The term "metal nanoprobe" used in the present invention is also used as a term such as "metal nanoparticle" or "metal nanosphere". The metal nanoprobe refers to a metal nanostructure, and is a term widely used in the art.

The term "magnetic particles" used in the present invention is also used as the term "magnetic beads". The magnetic particles may be composed of a magnetic material. As the magnetic material, any one can be used as long as it is widely used in the art. For example, one of Fe ₂ O ₃ , Fe ₃ O ₄ or FePt may be used, but is not limited thereto.

Magnetic particles and metal nanoprobes (eg, gold nanoparticles) used in the present invention are well known in the art (H. Chon, et. Al., Chem. Commun ., 2011, 47, 12515-12517 and Frens) , 1973, Nature Physical Science 241, 20-22., Etc.).

The Raman reporter is fixed to the surface of the metal nanoprobe. Since the Raman reporter shows a specific Raman spectrum, the target material to be detected in the SERS analysis can be analyzed more effectively. Any such Raman reporter molecule may be used as long as it is known in the art. Specifically, for example, 4,4'-dipyridyl (DP), crystal violet (CV), 4-mercapto toluene (4-MT), 3,5-dimethyl benzenethiol (3,5-DMT), Thiophenol (TP), 4-amino thiophenol (4-ATP), benzenethiol (BT), 4-bromo benzenethiol (4-BBT), 2-bromo benzenethiol (2-BBT), 4-iso Propyl benzenethiol (4-IBT), 2-naphthalene thiol (2-NT), 3,4-dichloro benzenethiol (3,4-DCT), 3,5-dichloro benzenethiol (3,5-DCT), 4 -Chloro benzenethiol (4-CBT), 2-chloro benzenethiol (2-CBT), 2-fluoro benzenethiol (2-FBT), 4-fluoro benzenethiol (4-FBT), 4-methoxy benzene Thiol (4-MOBT), 3,4-dimethoxy benzenethiol (3,4-DMOBT), 2-mercapto pyrimidine (2-MPY), 2-mercapto-1-methyl imidazole (2-MMI) , 2-mermetho-5-methyl benzimidazole (2-MBI), 2-amino-4- (trifluoromethyl) benzenethiol (2-ATFT), benzyl mertantan (BZMT), benzyl disulfide (BZDSF) , 2-amino-4-chloro benzenethiol (2-ACBT), 3-mercetobenzoic acid (3-MBA), 1-phenyltetrazol-5-thiol (1-PTE T), 5-phenyl-1,2,3-triazole-3-thiol (5-PTRT), 2-iodoaniline (2-IAN), phenyl isothiocyanate (PITC), 4-nitrophenyl disulfide (4-NPDSF), 4-azido-2-bromoacetophenone (ABAPN), X-rhodamine-5- (and-6) -isothiocyanate (XRITC), Crystal violet (CV) or malachite green isothiocyanate (MGITC) and the like, but are not necessarily limited thereto.

In an embodiment of the present invention, when using the gold nanoparticles described above, malachite green isothiocyanate (MGITC) may be used as the Raman reporter. According to an embodiment of the present invention, the Raman reporter molecule may be adsorbed onto the surface of the nanoprobe and immobilized by mixing the above-described metal nanoprobe.

The sex hormone detection antibody includes a first antibody and a second antibody, wherein the second antibody is immobilized on the magnetic particle, and the first antibody binds to the second antibody and reacts with the sex hormone. have.

The first and second antibodies may be used without limitation as long as they are used in the art. Specifically, the second antibody is an anti-mouse antibody immobilized on the magnetic particles; The first antibody may be an anti-sex hormone antibody that binds to the second antibody and specifically immune to the sex hormone.

The conventional immunoassay mainly used the "sandwich immunocomplex". The "sandwich immunocomplex" means an immunocomplex bound through an antibody-antigen-antibody reaction. It is named because the antigen is inserted in the middle of the antibody to form a sandwich.

To use the sandwich immunoassay, two antibodies must bind to the target (antigen) in the form of a sandwich. However, the sex hormones (antigens) of the present invention are small molecules. Sex hormones (antigens) do not have two epitope binding sites to which two antibodies can bind in the form of a sandwich of “antibody-antigen (sex hormone) -antibody”. Therefore, in the present invention, a competitive immune response (described later) was used to enable assay using one epitope binding site of sex hormone. In addition, when immobilizing the sex hormone antibody on the magnetic particles, in order to increase the loading density of the antibody (second antibody) was first immobilized, and then sex hormone-specific first antibody was immobilized again.

The sex hormone may be estrogen or testosterone, but is not necessarily limited thereto.

The detectable concentration of the sex hormone may be 0.1 to 1,000 pg / mL. Specifically, the detectable concentration of the sex hormone is preferably 0.1 to 10 pg / mL.

If the concentration of sex hormone is more than 1,000 pg / mL, for example, if the 3,000 pg / mL, it can be used diluted to 1,000 pg / mL.

The limit of detection of the sex hormone may be 0.1 pg / mL.

The sex hormone detection time may be 2 hours or less. Specifically, it may be 1 to 2 hours.

The estrogen may be selected from one or more selected from the group consisting of estradiol, estrone, and estriol, but is not necessarily limited thereto. The estradiol may be 17β-estradiol (E2).

The estrogen is a female hormone synthesized from cholesterol in the body and secreted into the ovary, the adrenal cortex, etc., and is a term for estradiol, estrone, and estriol. Estrogens play an important role in physiology such as oocyte maturation, mammary gland growth and development by binding to receptors in the nucleus and inducing female-specific sexual characteristics.

Estradiol is a sex hormone found mainly in women and is the most representative of the estrogens. Estradiol is known to affect the genital changes of the uterus, vagina, fallopian tubes, testicles, breast development, and induce fat dispersion and affect growth disorders. Estradiol levels in women ahead of childbirth are above 60 pg / mL and women receiving ovulation inducers have significant levels of estradiol (250-2000 pg / mL). Estradiol in men, children before puberty, and postmenopausal women is less than 20 pg / mL. In addition, estradiol levels in women with breast cancer administered sex hormone inhibitors are below 1 pg / mL.

Conventional diagnostic techniques have made it difficult to detect sex hormones below 10 pg / mL with high sensitivity. The present invention solves this problem and provides reagents that can detect sex hormones below 10 pg / mL with high sensitivity. The present invention also provides a detection reagent for which the detection time of sex hormone is 2 hours or less.

In another aspect, the present invention provides a SERS-based sex hormone detection method comprising the following steps:

Preparing a sample solution containing sex hormones;

Preparing a metal nanoprobe to which the sex hormone and Raman reporter are combined;

Simultaneously adding the metal nanoprobe, the magnetic particles to which the first antibody and the second antibody are immobilized, to the sample solution;

The sex hormone in the sample solution and the sex hormone of the metal nanoprobe each compete with the first antibody immobilized on the magnetic particles to form an immune complex with the magnetic particles;

Separating the magnetic particles on which the immunocomplex is formed by using magnetic;

Irradiating laser light on the separated magnetic particles; And

Detecting the sex hormone by measuring a Surface-Enhanced Raman Scattering (SERS) signal after the laser light irradiation.

As mentioned above, conventionally, "sandwich immunocomplexes" were mainly used. However, in the present invention, a "competitive immune response" in which an antibody immobilized on a magnetic particle reacts competitively with an antigen (sex hormone) bound to a metal nanoprobe and an antigen (sex hormone) present in a patient's blood (sample solution) Was used. That is, the present invention shortens the diagnosis time by simplifying the diagnosis procedure without forming a sandwich immunocomplex.

The competitive immune response may include an immune response between a sex hormone (antigen) included in the sample solution and a magnetic particle to which an antibody specifically binding to the sex hormone is immobilized (forming the first immune complex of FIG. 2); The immune response (forming the second immune complex of FIG. 2) between the metal nanoprobe bound to the same antigen as the sex hormone contained in the sample solution and the magnetic particles to which the antibody specifically bound to the sex hormone is immobilized is competitive. It may be an immune response that occurs.

The sample solution is selected from the group consisting of tissue extract, cell lysate, whole blood, plasma, serum, saliva, ophthalmic fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and dried blood spot However, it is not necessarily limited thereto. In the case of the blood filter, a sample solution may be prepared and used by a method well known in the art. Specifically, an extract obtained by extracting blood from blood spots may be used.

The second antibody is an anti-mouse antibody immobilized on the magnetic particle; The first antibody may be an anti-sex hormone antibody that binds to the second antibody and specifically immune to the sex hormone.

In the step of forming the immune complex, the higher the concentration of the sex hormone in the sample solution, the amount of the metal nanoprobe forming the magnetic particles and the immune complex decreases the SERS signal strength is measured; And / or the lower the concentration of sex hormone in the sample solution, the amount of the metal nanoprobe to form the immunocomplex with the magnetic particles may increase the SERS signal strength can be measured.

Figure 2 schematically shows a sex hormone detection method according to the present invention.

Preparing a sample solution containing sex hormones; Preparing a metal nanoprobe to which the sex hormone and Raman reporter are combined; Then, the magnetic particles to which the first and second antibodies for detecting sex hormone are immobilized are prepared. The metal nanoprobe combined with the sample solution and the Raman reporter is simultaneously added to the magnetic particles to which the first antibody and the second antibody are immobilized. With respect to the first antibody immobilized on the magnetic particles, a competitive immune response is induced between the sex hormone in the sample solution and the metal nanoprobe to which the Raman reporter (and sex hormone) is bound to form an immunocomplex. At this time, the first immunocomplex in which the sex hormone in the sample solution and the first antibody of the magnetic particles are bound, and the second immunocomplex in which the sex hormone of the metal nanoprobe and the first antibody of the magnetic particles are bound at the same time are formed. .

After the immunocomplex is formed, the magnetic particles on which the immunocomplex is formed are separated using magnetism; The surface-enhanced Raman Scattering (SERS) signal is measured by irradiating the separated magnetic particles with laser light.

If the concentration of sex hormone in the sample solution is high, a small amount of the second immunocomplex to which the metal nanoprobe to which the Raman reporter is bound and the first antibody to the magnetic particles are bound will be produced. It demonstrates concretely with FIG. FIG. 4 is a transmission electron microscopy (TEM) image of an immunocomplex formed through the competitive immune response (referred to as an organic substance, sex hormone is not observed by TEM). 4 is a concentration of estradiol, a sex hormone, in the sample solution. As can be seen from Figure 4, it can be seen that the higher the concentration of sex hormone in the sample solution, the amount of the metal nanoprobe binding to the magnetic particles is reduced. The concentration of metal nanoprobe used in the competitive immune reaction of the present invention is optimized for testing. The concentration of the metal nanoprobe used in one embodiment of the present invention is 0.12 nM, when inspecting a sample of 25 μL, 50 μL of metal nanoprobe was used. In other words, the amount of metal nanoprobe used in the competitive immune reaction is constant. If the concentration of sex hormones in the sample solution is high, the sex hormones in the sample solution are resistant to the sample solution when the immune response competes with the sex hormones of the metal nanoprobe with the magnetic particles (fixed) in between. The hormones will be more likely to combine with the magnetic particles to form an immune complex. As a result, the amount of metal nanoprobe attached to the magnetic particles will be less (ie, the amount of the second immunocomplex is less than the amount of the first immunocomplex), and thus the strength of the SERS signal will be weakly measured. On the contrary, if the sex hormone concentration in the sample solution is low, the first immunocomplex of the sex hormone and the magnetic particles in the sample solution will be less formed. In addition, the amount of the second complex in which the metal nanoprobe and the first antibody of the magnetic particles are bound will be relatively large. In this case, since the metal nano probe is attached to the magnetic particles, the intensity of the SERS signal will increase.

Therefore, if the concentration of the sex hormone in the sample solution is high, the intensity of the SERS signal can be measured low, and if the concentration of the sex hormone in the sample solution is low, the SERS signal strength can be measured high.

In another aspect, the present invention,

Extracting the sample liquid from the subject;

Sex hormones are detected in the extracted sample liquid through the above-described sex hormone detection method; And

Confirming the concentration of the detected sex hormone;

It provides a method for providing information necessary for diagnosing precocious puberty, including.

The subject is preferably a mammal, specifically a human, and more preferably a female child under 8 years old or a male child under 9 years old.

The concentration of the detected sex hormone may be 0.1 to 1000 pg / mL.

After the step of confirming the concentration of the detected sex hormone, when the concentration of the detected sex hormone is more than 10 pg / mL, may further comprise the step of diagnosing that the subject providing the sample solution is precocious puberty .

Sex precocious puberty can be diagnosed using a reagent kit for detecting sex hormones and a method for detecting sex hormones according to the present invention. In addition, since the present invention can detect a very low concentration of sex hormone, the reagent kit and sex hormone detection method according to the present invention can be used for various indications related to sex hormone.

For example, the present invention can be applied to the setting of a normal range of estradiol according to age and gender in children, tracking the onset of pubertal development, prognosis by diagnosing precocious puberty and determining drug effects after treatment of precocious puberty. have. In addition, the prognosis of pharmacotherapeutic effects (estradiol should be measured below 1 pg / mL in breast cancer patients), risk factors for osteoporosis and fractures in men and gonadal tumors in breast cancer patients undergoing hormonal therapy The present invention can be applied to diagnosis, to finding the cause of a boy having a feminized breast. The present invention can also be applied to the diagnosis and prognosis of hormone therapy in postmenopausal women, determination of the effects of hypogonadism, determination of hormonal drug effects in patients with prostate cancer, and the like.

By using the sex hormone detection reagent and detection method according to the present invention, very small concentrations of sex hormone can be detected. Therefore, using the present invention, accurate diagnosis of precocious puberty is possible. In addition, the present invention can detect sex hormones with high sensitivity, and thus may be used for the study of hormone-related diseases.

Figure 3 is a transmission electron microscope (Transmission Electron Microscope, TEM) image (a) and dynamic light scattering (DLS) measurement analysis results of the synthesized gold nano probe (b).

Figure 4 is a TEM image of an immunocomplex formed through a competitive immune response according to the present invention (concentration value in the upper left of the image is the estradiol concentration in the sample solution).

5a to 5c are graphs showing a Raman signal measurement result through a method for detecting estradiol according to an embodiment of the present invention.

6A to 6C are graphs showing results of measuring Raman signals through a testosterone detection method according to an embodiment of the present invention.

7 is a graph showing the estradiol detection results by the SERS-based detection method according to an embodiment of the present invention, and the estradiol detection results through ELISA analysis as a comparative example.

8 is a graph showing testosterone detection results through ELISA analysis as a comparative example.

Hereinafter, preferred examples are provided to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications can be made within the scope and spirit of the present invention. It is obvious to the skilled person, and it is natural that such variations and modifications fall within the scope of the appended claims.

Example 1 Preparation of First Reagent--Metal Nanoprobe Preparation

Gold chloride (HAuCl ₄ ), Trisodium citrate, poly (ethylene glycol) 2-mercaptoethyl ether acetic acid (HS-PEG-COOH, MW-3500), poly (ethylene glycol) methyl ether thiol (HS-PEG, MW-2000), EDC (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) are from Sigma-Aldrich, malachite green isothiocyanate (MGITC) is from Invitrogen, and estradiol-ofalbumin (Estradiol-ovalbumin conjugate, E2-OVA) was purchased from Cusabio.

To synthesize metal nanoprobes, spherical gold nanoparticles were synthesized (Frens, 1973, Nature Physical Science 241, 20-22.). After boiling 50 mL of a 0.01% solution of HAuCl ₄ , 0.5 mL of a 1% solution of trisodium citrate was added dropwise. The initial HAuCl ₄ aqueous solution turned blue when the nanoparticles were formed, and gradually changed to red as the nanoparticles grew over time. Finally, after confirming the color of the nanoparticles to be synthesized, the reaction was terminated by further boiling for 15 minutes. After the reaction was completed, the temperature of the gold nanoparticles was lowered to room temperature, and subjected to aging for 4 hours or more. As shown in FIG. 3, the synthesized gold nano probe was confirmed to be uniformly and stably synthesized in a size of about 40-50 nm through an electron transmission microscope (TEM) and light scattering (dynamic light scattering) measurement. Then, malachite green isothiocyanate (MGITC), which is a Raman reporter, was coated on the gold nanoparticles to use as a SERS substrate. Raman reporter was added dropwise to a final concentration of 50 nM to 1 mL of 0.12 nM 40-nm gold nanoparticles, and then 10 μM poly (ethylene glycol) 2-mercaptoethyl ether acetic acid ( 60 μL of HS-PEG-COOH, MW ˜3500) and 120 μL of 10 μM poly (ethylene glycol) methyl ether thiol (HS-PEG, MW ˜2000) were added dropwise and reacted for 3 hours. Thereafter, the carboxyl functional groups on the surface of the gold nanoparticles were used to immobilize Estradiol-ovalbumin conjugate (E2-OVA) or testosterone-bovine serum albumin conjugate (BSA), and for this, 25 mM EDC (N 5 μL of-(3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) were added dropwise, followed by reaction for 15 minutes, followed by 2 μL addition of 1 mg / mL estradiol-ofalbumin and 2 hours at room temperature. After the reaction, the reaction was carried out at 4 ° C. for 12 hours. Thereafter, the unreacted residue on the surface of the gold nanoparticles was removed by centrifugation (7200 rpm, 10 minutes).

Example 2 Preparation of Second Reagent-Manufacture of Magnetic Particles

The second antibody (anti-mouse antibody, anti-Mouse IgG (Fc specific) antibody produced in goat), EDC (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) are Sigma-Aldrich In the micro-magnetic particles (Dynabeads MyOne ^™ ), PBS buffer (0.1 mM, pH 7.4), Invitrogen, the first antibody [(anti-estradiol antibody, anti-17 beta Estradiol antibody) or (anti-testosterone antibody, mouse anti-testosterone monoclonal antibody) was purchased from Abcam.

The second antibody (anti-mouse antibody) was immobilized using a carboxyl functional group on the surface of the micro magnetic particles. To this end, the carboxyl functional groups on the surface of the micro magnetic particles were activated for 30 minutes by adding 5 M of 0.1 M EDC and NHS dropwise. Thereafter, 2.5 mg of 2 mg / mL secondary antibody (anti-mouse antibody) was added dropwise and reacted at room temperature for 2 hours. Then, magnetic particles were separated and magnetic residues were removed to remove residues that did not react on the surface of the micro magnetic particles. The solution was dissolved in PBS (10 mM, pH 7.4) and stored at 4 ° C. Subsequently, 25 μL of the anti-estradiol antibody (or anti-testosterone antibody), which is 0.55 g / mL of the first antibody, is added dropwise to 25 μL of the magnetic particle having the second antibody fixed therein, and reacted at room temperature for 90 minutes to detect sex hormones. The preparation was completed. After the reaction, the magnetic particles were separated by magnetic force to remove the unreacted residue on the surface of the micro magnetic particles, and the residue was prepared by dissolving the residue in PBS (10 mM, pH 7.4).

Example 3: Sex Hormone Detection Based on Surface Enhanced Raman Scattering Technology

3-1: Estradiol detection

First, blood containing estradiol was prepared as a sample solution. Next, 25 μL of the magnetic particles having the second antibody synthesized in Example 2 and the first antibody (anti-estradiol antibody) immobilized on 25 μL of the sample solution and 50 μL of the gold nanoprobe prepared in Example 1 simultaneously. The total test time for addition was 90 minutes.

Figure 4 is a transmission electron microscopy (TEM) image of an immunocomplex formed through a competitive immune response according to the present invention (concentration value in the upper left of the image is the estradiol concentration in the sample solution). As can be seen from Figure 4, it can be seen that the higher the concentration of sex hormone in the sample solution, the amount of the metal nanoprobe binding to the magnetic particles is reduced.

Then, magnetic particles in which the first and second immunocomplexes were formed were separated using magnetic force, and Raman analysis was performed on the separated magnetic particles. Raman analysis was performed as follows. Renishaw Invia Raman spectrometers (Renishaw, UK) were used, and a Spectra Physics He-Ne 632.8 nm laser (Spectra Physics He-Ne 632.8 nm laser) was used as the light source. The Rayleigh line was removed using a haloographic notch filter located in the collection path. The spectra were corrected for peak position by measuring the silicon reference peak position at 520 cm ⁻¹ before the measurement. Raman spectra were collected in the 630-1730 cm ^-1 region with a 1 second acquisition time using a laser with an output wavelength of 633 nm and an output intensity of 20 mW. A 20 x objective was used to focus the laser spot. Baseline correction of all spectral data was adjusted with WiRE 4.0 (Renishaw, UK) software. The quantitative results of sex hormones were analyzed based on the 1613 cm ^-1 signal intensity, the strongest peak of the Raman reporter used.

As a result, as shown in Figures 5a to 5c, the Raman signal results were shown. The minimum detectable concentration was found to be 0.1 pg / mL.

3-2: Testosterone Detection

First, blood containing testosterone was prepared as a sample solution. Then, 25 μL of the magnetic particles immobilized with the second antibody and the first antibody (anti-testosterone antibody) synthesized in Example 2 and 50 μL of the gold nanoprobe prepared in Example 1 were simultaneously added to 25 μL of the sample solution. The total test time was 90 minutes.

Raman analysis was performed using the Raman analysis method disclosed in Example 3-1. As a result, as shown in Figures 6a to 6c, the Raman signal results were shown. The minimum detectable concentration was found to be 0.1 pg / mL.

Comparative example : Comparison with ELISA Diagnostic Methods

One: Estradiol ELISA analysis for detection

Enzyme immunoassay (ELISA) is a general medical diagnostic analysis method in which the blood injected with antigen is judged to be the action of an enzyme causing color change. In order to verify the sensitivity of the sex hormone analysis technique using the surface enhanced Raman spectroscopy according to the present invention, it was compared with the results analyzed using the estradiol detection kit (ELISA method) of Abnova. This diagnostic method is a quantitative analysis of a competitive reaction between estradiol and luminescent inducers labeled estradiol. This diagnostic method conjugated an antibody capable of immobilizing estradiol on an assay plate, placed an analytical sample, and allowed the antibody conjugated with a luminescent inducer to conjugate to an immobilized antigen. Diagnosis was confirmed by measuring the degree of color change by the device according to the content of the luminous inducing material. Substances provided for analysis are shown in Table 2 below.

TABLE 2

Figure 7 compares the surface enhanced Raman scattering based sex hormone detection results (a) and ELISA analysis results (b). Comparing the two detection methods, the SERS based detection method according to the present invention has a detection range of 0.1 to 1,000 pg / mL and a detection limit of 0.1 pg / mL. In contrast, ELISA assays have a detection range of 5-1,000 pg / mL and a detection limit of 5 pg / mL.

2: ELISA assay for testosterone detection

The materials provided for ELISA analysis are shown in Table 3 below.

TABLE 3

8 compares the results of ELISA analysis of testosterone. Compared with the SERS based detection method of the present invention, the SERS based detection method according to the present invention has a detection range of 0.1 to 1,000 pg / mL and a detection limit of 0.1 pg / mL. In contrast, ELISA assays have a detection range of 1-100 ng / mL and a detection limit of 0.18 ng / mL (= 180 pg / mL).

From this result, it can be seen that the SERS-based detection method according to the present invention can also analyze samples in a concentration range of 0.1-5 pg / mL that cannot be analyzed by ELISA. In particular, for the diagnosis of precocious puberty it is necessary to detect a low concentration sex hormone sample less than 10 pg / mL, the present invention satisfies this demand. However, ELISA assays cannot detect low concentrations of sex hormones below 10 pg / mL. The results shown in FIG. 6 show that the SERS-based detection method according to the present invention is an analysis method capable of detecting high sensitivity necessary for diagnosing precocious puberty.

Example 4: Clinical Effectiveness Assessment

Blood of 30 real patients was analyzed using ARCHITECT's estradiol analysis method (automatic analysis). ARCHITECT's estradiol detection method is an immunoassay method using a chemiluminescent material is a method capable of quantitative analysis from the chemiluminescence signal according to the amount of sex hormone present in the blood. The detectable range of this method is 10-1000 pg / mL and the detection limit is 10 pg / mL, so no analysis results are provided for samples below 10 pg / mL. Table 4 shows the test results of blood hormone levels in 30 patients who visited the hospital with an estradiol analyzer. In addition, Table 4 analyzes the same blood sample using the SERS-based detection method according to the present invention and compares the results. Looking at the analysis results of Table 5, it can be seen that the SERS-based detection results show very significant results with the ARCHITECT's estradiol detection method for all 30 blood. In particular, it can be seen that accurate results can be analyzed even for samples less than 10 pg / mL that were not provided in the estradiol detection result of ARCHITECT. Through this, the clinical effectiveness of the SERS-based detection method according to the present invention has been sufficiently verified. In particular, it can be seen that the analysis performance of low-concentration sex hormone samples less than 10 pg / mL is also superior to the existing hematological analysis equipment. Therefore, it can be seen that the SERS-based detection method according to the present invention is a very suitable method for diagnosing precocious puberty by detecting high sensitivity sex hormone in blood.

TABLE 4

TABLE 5

Claims

A first reagent comprising a metal nanoprobe to which a sex hormone and a Raman reporter are immobilized; And

A second reagent comprising magnetic particles immobilized with the sex hormone detection antibody;

Reagent kit for detecting sex hormone comprising a.
The method of claim 1,

The sex hormone detection antibody comprises a first antibody and a second antibody, the second antibody is immobilized on the magnetic particles, the first antibody binds to the second antibody and reacts with the sex hormone Reagent kit for detecting sex hormones.
The method of claim 1,

The sex hormone is a reagent kit for detecting sex hormone estrogen or testosterone.
The method of claim 3,

The estrogen is one or more selected from the group consisting of estradiol, estrone (estrone) and estriol (estriol) reagent kit for detecting sex hormone.
The method according to any one of claims 1 to 4,

Reagent kit for detecting sex hormone is the detectable concentration of the sex hormone is 0.1 to 1,000 pg / mL.
The method according to any one of claims 1 to 4,

The limit of detection of the sex hormone (limit of detection) is 0.1 pg / mL reagent kit for detecting sex hormone.
The method according to any one of claims 1 to 4,

The sex hormone detection time is 2 hours or less reagent kit for detecting sex hormone.
Preparing a sample solution containing sex hormones;

Preparing a metal nanoprobe to which the sex hormone and Raman reporter are combined;

Preparing magnetic particles to which the first and second antibodies for detecting sex hormone are immobilized;

Simultaneously adding the metal nanoprobe, the magnetic particles to which the first antibody and the second antibody are immobilized, to the sample solution;

The sex hormone in the sample solution and the sex hormone of the metal nanoprobe each compete with the first antibody immobilized on the magnetic particles to form an immune complex with the magnetic particles;

Separating the magnetic particles on which the immunocomplex is formed by using magnetic;

Irradiating laser light on the separated magnetic particles; And

Detecting the sex hormone by measuring a surface-enhanced Raman Scattering (SERS) signal after the laser light irradiation;

SERS-based sex hormone detection method comprising a.
The method of claim 8,

In the step of forming the immunocomplex,

As the concentration of sex hormone in the sample solution increases, the amount of metal nanoprobes forming the immunocomplex with the magnetic particles decreases, so that the SERS signal strength is lowered; or

The lower the sex hormone concentration in the sample solution, the greater the amount of metal nanoprobe to form the magnetic complex with the magnetic particles, the higher the SERS signal strength detection method based on SERS.
The method of claim 8,

The sex hormone detection method based on SERS is estrogen or testosterone.
The method of claim 10,

The estrogen is one or more selected from the group consisting of estradiol, estrone (estrone) and estriol (estriol) SERS-based sex hormone detection method.
The method of claim 8,

The sample solution is selected from the group consisting of tissue extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and dried blood spot SERS-based sex hormone detection method.
The method of claim 8,

The second antibody is an anti-mouse antibody immobilized on the magnetic particle; The first antibody is an SERS-based sex hormone detection method that binds to the second antibody and is an anti-sex hormone antibody that specifically reacts with the sex hormone.
The method according to any one of claims 8 to 13,

The detectable concentration of the sex hormone is 0.1 to 1,000 pg / mL SERS based sex hormone detection method.
The method according to any one of claims 8 to 13,

The limit of detection of the sex hormone (limit of detection) is 0.1 pg / mL SERS-based sex hormone detection method.
The method according to any one of claims 8 to 13,

The sex hormone detection time is less than 2 hours SERS-based sex hormone detection method.
Extracting the sample liquid from the subject;

Sex hormone is detected from the extracted sample liquid through the sex hormone detection method according to claim 8; And

Confirming the concentration of the detected sex hormone;

The method for providing information necessary for diagnosing precocious puberty of a subject.
The method of claim 17,

The concentration of the detected sex hormone is characterized in that 0.1 to 1000 pg / mL.
The method of claim 17,

After confirming the concentration of the detected sex hormones,

If the concentration of the detected sex hormone is more than 10 pg / mL, further comprising the step of diagnosing precocious puberty.